Search

The primary goal of the project is to develop molecules with the potential to alleviate the symptoms and slow down the progression of neurodegenerative diseases that primarily affect the neurons in the human brain that causes problems with movement and/or mental functioning. As the best results in restoration of cognitive functions of patients and alleviating the symptoms of the disease are done using drugs that targets cholinesterases (ChE), the project aims to rationally design dual site binding ChE inhibitors (acting on improving the acetylcholine level in the brain and on Aβ aggregation) and use them as starting points for multitarget-directed ligand (MTDL) design. We focused our study on butyrylcholinesterase (BChE) selective inhibitors due to the role of the BChE in the regulation of brain ACh levels in late AD and the fact that selective inhibition of BChE reduces the occurrence of side effects seen with the acetylcholinesterase (AChE) or nonselective ChE inhibitors currently in use. In design of potential bioactive molecules, we chosen two structural scaffolds, each with different mode of action with ChE’s. A carbamate functionality was chosen due to the similarity of mechanism of their interaction with choliesterases with the mechanism of AChE hydrolysis of its physiological substrate ACh. Aminoquinoline, as a structural motive, gained our attention due to their similarity to tacrine, the first centrally acting cholinesterase inhibitor approved for the treatment of Alzheimer’s disease.

In a recent study, quinuclidine-based carbamates showed significant anti-acetylcholinesterase and anti-butyrylcholinesterase activity, making them promising central nervous system agents [1]. Binding modes of selected quinuclidine derivatives within cholinesterase’s active site were investigated by Monte Carlo quantum chemical docking. The configurational space of each ligand was spanned in 3 translational, 3 rotational, as well as all relevant torsional degrees of freedom and sampled using an implemented stochastic search algorithm [2]. The protein was kept rigid during the docking process while small molecules were allowed to explore the active site. For every generated configuration, single-point calculations were performed using the semiempirical PM7 Hamiltonian. Lowest-energy Michaelis complexes were singled out and optimized at a higher level of theory. Combined QM/QM optimizations were conducted using the ONIOM method and relative standard Gibbs energies of binding were calculated. All quantum chemical calculations were performed using the Gaussian 16 program package [3].

Guided by an interest to develop new cholinesterase-acting drugs with better biological activity and bioavailability, we have synthesized 14 quaternary quinuclidine compounds with the variation in N-alkyl chain length and incorporation of alcohol or oxime headgroup at the position 3 of the quinuclidine ring. SwissADME interface was used to determine basic physicochemical properties and to evaluate whether tested compounds interact with selected enzymes of the cytochrome P450 system, which could affect their metabolism. Most of the tested quinuclidines complied with the Lipinski's rule-of-five ensuring their drug-likeness properties. Exceptions were noticed for bisquaternary 3-hydroxy and 3- hydroxyimino compounds with C8 and C10 alkyl chains which exceed the number of rotating bonds and topological polar surface area compared to the recommended values. Furthermore, it was estimated that all compounds except 3-hydroxyimino compound with C16 alkyl chain have an ability to cross the blood brain barrier and favorable properties for gastrointestinal absorption. However, all tested quinuclidines have either negative logP value or below 1.5, indicating low lipophilicity and therefore low potential to be passively transported across biological barriers. According to computer prediction quinuclidines with a long side alkyl chain (C12 and C14) inhibit CYP2D6 that may be one of the causes of pharmacokinetics- related drug-drug interactions leading to toxic or other unwanted adverse effects. Understanding how combination of different functionalities in a molecule affects its physicochemical properties represents a good starting point for further biological research of activity and toxicity.

Spectroscopic measurements of 82 selected odorants were performed using 1H NMR spectroscopy. This set includes 6 main types of perfumery odor notes[1] and the NMR spectral data will subsequently be used to build an accurate classification model. 2nd- order tensor decomposition tool principal component analysis (PCA) was applied to a set of obtained NMR spectra, as well as on their first and second derivatives. The quality of PCA models was evaluated by determining the optimal number of principal components for the representation in the reduced space.[2] In each case, the first principal component accounted for most of the total variance among the samples. The results were additionally improved using spectral derivatives. Classification of these odorants was established and underlying hidden spectral differences among compounds were determined by investigating the principal component loadings.[3] These differences are directly caused by changes in the chemical composition. It was found that NMR spectroscopy coupled with PCA can distinguish between various fragrant compounds. Odorants subjected to the chemometric analyses can be divided into several major groups (clusters). Investigation of the principal component loadings determined the major differences among the NMR spectra regarding structural patterns present in the chemical structures. These differences are associated with the total number of aromatic and/or aliphatic functional groups and their structure, reflecting variations in the composition of different odor notes.

Quinuclidine based derivatives are a class of compounds that has been attracting increased attention in modern drug discovery based on their anticholinergic, antimicrobial, antioxidative and antitumor activity. Guided by an interest to develop drugs with polypotent chemical structures which result in interaction with various molecular targets or receptors, we have synthesized and characterized 14 quaternary quinuclidine compounds with the variation in N-alkyl chain length and incorporation of alcohol or oxime headgroup at the position 3 of the quinuclidine ring. We analysed reversible inhibition of human acetylcholinesterase (AChE) and human butyrylcholinesterase (BChE) by these compounds and screened their cytotoxic effect on three selected cell types: neuronal cells (SH-SY5Y), hepatocytes (Hep G2) and kidney cells (HEK293). All of the tested compounds reversibly inhibited both AChE and BChE activity in the micromolar range. The most potent inhibitors were bisquaternary 3-hydroxy and 3-hydroxyimino compounds with C10 alkyl chains (Ki = 0.2 – 1.6 µM). Analysis of the impact of functional group revealed oximes with C8 to C12 alkyl chains and alcohols with C14 and C16 alkyl chains as more potent reversible inhibitors than their analogues. AChE showed high affinity toward compounds with a long alkyl side chain (C14 and C16). BChE affinity was not affected much by the length of the alkyl chain, although an opposite preference to AChE was observed. MTS assay showed toxicity (IC50 = 3 – 204 µM) of compounds with a long side alkyl chain (C12, C14 and C16) from both groups, alcohols and oximes. Neuronal cells were the least sensitive to the toxicity of the tested compounds. According to inhibition potency for both AChE and BChE and non- cytotoxic profile, it seems that quinuclidine oximes with short chain length (< C12) can be considered as candidates for further investigations with possible therapeutic potential in central nervous system.

Cinchonidine belongs to class of alkaloids found in the bark of Cinchona tree, together with cinchonine, quinine and quinidine. These alkaloids have various uses in chemistry, for example they are used as organocatalysts in asymmetric synthesis or as chiral stationary phases and chiral solvating agents in chromatographic separations. Also, they posses a wide range of biological activity such as antimalarial, antibiotic and antiparasitic. By transfer hydrogenation of cinchonidine using formic acid/ammonium formate as hydrogen donor and palladium on carbon as a catalyst 10, 11 dihydrocinchonidine was prepared and used as starting material for synthesis of his carbamate derivatives. We have prepared a series of mono- and disubstituted aliphatic (methyl, ethyl) and aromatic (phenyl) 10, 11 dihydrocinchonidine carbamates. Carbamate derivatives have been prepared by reaction of 10, 11 dihydrocinchonidine with appropriately substituted isocyanates or carbamoyl chlorides. Synthesized compounds were characterized by FT-IR, 1D and 2D 1H and 13C NMR spectroscopy. Extensive docking studies and quantum chemical calculations were used to define intermediates and transition states in the course of the carbamylation reaction as well as other possible interactions of prepared compounds within the active sites of enzymes. All prepared carbamate derivatives will be screened for their ability to inhibit human acetylcholinesterase and butyrylcholinesterase activity.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are enzymes crucial for neurotransmitter acetylcholine metabolism in both central and peripheral nervous system. The progressive inhibition of these enzymes by nerve agents (NA) during poisoning disrupts acetylcholine breakdown, leading to excessive impulse transmission and cholinergic crisis. Since BChE circulates freely in the bloodstream, it has been considered as a possible exogenous bioscavenging enzyme which would act as an oxime- assisted catalytic scavenger of NAs, neutralising them before they reach target tissues. Oximes are used in standard clinical therapy to recover activity of inhibited cholinesterases, but reactivating BChE with standard pyridinium oximes proved to be ineffective as they are mainly AChE reactivators. To approach this issue, based on previous research we synthesized eight novel N- benzyl supstituted imidazolium oximes to be considered as reactivators of nerve agent sarin-, cyclosarin- and tabun-inhibited human AChE and BChE. Two oximes emerged as effective reactivators of all three NA-BChE conjugates, possessing superior overall reactivation rates (kr) than standard oxime HI-6 which was up to 3000-times higher for cyclosarin conjugate and 210-times for sarin conjugate, or 100-times higher than oxime TMB-4 in case of tabun conjugate. Binding affinities of phosphylated BChE for novel oximes in terms of 1/KOX increased up to 1900-fold compared to standard oximes and they achieved their reactivation maximum in a shorter time frame. However, standard pyridinium oximes remain better reactivators of phosphylated AChE which indicates that imidazolium oximes with 1, 3- aromatic substituents do not position optimally into the AChE active site gorge. Novel oximes' low toxicity on hepatic (HepG2) and neuronal (SH-SY5Y) cell lines and high capacity for reactivation of BChE in the studied concentration range establishes them as promising candidates for future treatment development.

Antidotes for organophosphorus poisoning are oximes, which antidotal properties are related to their ability to reactivate phosphorylated acetyl- cholinesterase (AChE, EC 3.1.1.7), and butyrylcholinesterase (BChE, EC 3.1.1.8) when used as scavenger [1]. In addition, it is well known that the protection of cholinesterases (ChEs) from phosphorylation can be achieved by the previous inhibition with carbamates. Given that there is no single compound applicable as antidote for poisoning with various organophosphorus agents, pursue for compounds with better chemical, physical and biochemical properties as well as better understanding of their interactions with both enzymes are still important. Therefore, over the years, a series of novel quinuclidine and cinchona-based oximes and carbamates were synthesized to be evaluated as inhibitors and/or reactivators of phosphorylated human ChEs [2, 3]. Compounds were modified to gain divers structures and the hybrid molecules with known aromatic pyridine and imidazole oximes designed. Activity and selectivity of cholinesterases were described, and to clarify differences in the inhibition and oxime reactivation potency, conformational analysis of compounds as well as detailed docking studies were conducted. Orientations of studied compounds in the active site of ChEs have been proposed by QM/QM studies. Analyses of the obtained complexes pointed out significant hydrogen bonds and close contacts between functional groups of compounds and the residues of the active site. To facilitate the prediction and design of new and more potent compounds, the most optimal regression models for the prediction of bioactivity were established and validated by extensive machine learning protocols. (Supported by the Croatian Science Foundation, Project No. IP-2016-06-3775 ADESIRE)

A series of 25 fluorinated Cinchona alkaloids derivatives was theoretically investigated by calculation of their potential energy surfaces (PES). PES for all compounds were sampled by performing molecular dynamics simulations [1] and then decomposed by principal component analysis. Each PES was represented by three points in the newly determined reduced space. These points were used as independent variables for establishing activity/PES regression models whereas previously measured inhibitory activities towards human acetyl- and butyrylcholinesterase were used as dependent variables. Multivariate linear regression models were built by applying an extensive machine learning protocol where linear combinations of original variables as well as their higherorder polynomial terms were used. Leave-one-out cross- validation (LOO-CV) was used to validate obtained models [2, 3]. Optimal activity/PES models were selected based on the adjusted R 2, predicted R 2 and the LOO-CV mean squared error (Figure 1).

Cinchona alkaloids are known natural products with multiple bioactivities Accordingly, it can be expected that compounds structurally related to Cinchona alkaloids might also possess various biological activities. To continue our work and additionally explor e Cinchona-alkaloid-derived compounds as cholinesterases inhibitors, 46 new quaternary derivatives of cinchonines and their corresponding pseudo-enantiomeric cinchonidines were synthesised and evaluated as human cholinesterases inhibitors Alkaloids were modified by quaternization of quinuclidine moiety of each compound with groups containing fluorine atoms Furthermore, to determine the role of the vinyl group in binding of compounds to ChEs, 10, 11- dihydrocinchonidine derivatives were prepared by transfer hydrogenation of cinchonidine using formic acid/ammonium formate as hydrogen donor All prepared compounds proved to be reversible inhibitors of human butyrylcholinesterase and acetylcholinesterase with Ki constants from nanomolar to micromolar range Cinchonidine derivatives displayed higher inhibition selectivity toward ChEs than cinchonines Three cinchonidine compounds were 84–533 times better inhibitors of butyrylcholinesterase than acetylcholinesterase Some cinchonidine derivatives were potent butyrylcholinesterase inhibitors with Ki constants up to 100 nM, of which N-meta- fluorobenzylcinchonidinium bromide can be considered as a lead for further modifications and optimizations of structure to obtain a higher butyrylcholinesterase inhibition potency and selectivity To enable the better prediction and facilitate design of new and more efficient inhibitors, the most optimal regression models for the prediction of bioactivity were established and validated by extensive machine learning protocol.

Jedan od rjeđe korištenih pristupa za sintezu oksima je i mehanokemijska sinteza [1] koja je privukla značajan interes istraživačke zajednice zbog mogućnosti provođenja kemijskih reakcija bez upotrebe toksičnih i/ili skupih otapala, ali i mogućnosti da se dobiju različiti glavni produkti takvim reakcijama u odnosu na one koje se izvode u otopini [2]. U okviru ovog rada bit će opisana priprava O - supstituiranih oksima kinuklidin-3-ona. U reakciji kinuklidin-3-ona ili kinuklidin-3-onhidroklorida te odgovarajućih O -supstituiranih hidroksilamin-hidroklorida pripravljeni su metilni, benzilni, tert -butilni i fenilom suptituirani oksimi kinuklidin-3-ona. Uspoređeni su rezultati sinteze u otopini i mehanokemijske sinteze koji ukazuju na prisutnost oksima (E ) i (Z ) konfiguracije. Obzirom na dobivene rezultate dodatno je istraženo kako promjena reakcijskih uvjeta i korištenja različitih baza utječe na omjere dobivenih stereoizomera. ATR mjerenjima tijekom eksperimenata praćena je kinetika kemijskih reakcija. Identifikacija i kontrola čistoće pripravljenih spojeva ispitana je klasičnim analitičkim postupcima koji su uključivali ATR i 1H NMR spektroskopiju.

Potential energy surfaces (PES) for 25 fluorinated Cinchona alkaloids derivatives were sampled by ab initio molecular dynamics [1] and used as independent variables in establishing activity/PES multivariate linear regression models (MLR) [2, 3]. Principal components of previously measured inhibitory activities towards human acetyl- and butyrylcholinesterase were used as dependent variables. An extensive machine learning protocol was applied for generating all possible MLR models with linear combinations of original variables as well as their higher-order polynomial terms. Evolution of regression model was monitored by calculation of R2, adjusted and predicted R2. Each regression model was fully validated by leave-one- out cross- validation (LOO-CV) and the best possible activity/PES models for different dimensionalities were selected based on R2 values and the LOO-CV mean squared errors (Fig. 1).

Listeriosis is a serious disease caused by food contaminated with Listeria monocytogenes. This bacterium is found in soil and surface water samples, but can also be found in dairy products, meat and seafood. Membrane-active antimicrobials such as quaternary ammonium salts (QASs), represent an important class of bacteriostatic and biocidal amphiphilic agents that are widely used in daily life. Given the widespread use of QASs, there have been reports of L. monocyogenes isolates resistant to these compounds. Here, we present the synthesis of a new series of quaternary amidoquinuclidine salts with potent antibacterial activity against L. monocytogenes. The antibacterial activity was determined for bacteria in the form of suspension and biofilm in the low micromolar range. The newly synthesized 3- aminoquinuclidines (C10, C12, C14, and C16) were quaternized with allyl or methyl substituents. We found that regardless of the type of substituent, the derivatives with longer alkyl chains had minimal inhibitory concentrations against L. monocytogenes that were almost identical to those of the standards (BAB, CPC, and cefotaxime). The identified candidates had low toxicity to healthy human cell lines with potent genotoxic activity against bacterial cells. In addition to the mechanism of action, we demonstrated that these agents target the cell membrane and cause membrane perforation and cell death. Parallel artificial membrane permeability assays mimicking human skin showed that candidates with longer alkyl chains did not penetrate the membrane, highlighting their potential application as disinfectants against this and other highly pathogenic bacteria.

In the self-condensation reaction of quinuclidin- 3-one, two distinct isomers are produced in different ratios. To explain this difference in isomer content, full conformational analyses of all 2-(3-hydroxyquinuclidin-3-yl)quinuclidin-3-one stereoisomers were performed. Conformational spaces were calculated by using grid search methods and tensor decomposition of ab initio molecular dynamics trajectories. Grid search was performed by rigid scan and a new adaptive relaxed scan method implemented in program msa at the semiempirical level of theory using PM7 Hamiltonian. Molecular dynamics simulations were performed using on-the-fly calculations of forces in each point of the simulation and velocity Verlet algorithm for integration implemented in program qcc. Principal component analysis was used as a dimensionality reduction tool and performed using a NIPALS algorithm implemented in program moonee. Complete conformational spaces for investigated compounds in the ground state have been determined and results obtained from each individual method have been compared. Use of principal component analysis on generated molecular dynamics trajectories provided the complete set of conformers for every investigated system contrary to the grid search methods, which excluded some of them. Full conformational spaces of enantiomeric pairs were determined with exact match between enantiomeric conformers confirming the validity of used methods and these results are used to explain the differences in self- condensation reaction products.

Bioaktivne molekule često sadrže neke zajedničke strukturne motive koji čine okosnicu njihovih molekularnih struktura [1]. Suvremeni putevi u sintezi biomimetskih lijekova koji sadrže neke od najčešćih malih bioaktivnih skeleta ilustriraju potencijal ovih molekula u istraživanjima novih mogućih lijekova. Neki odabrani bioaktivni skeleti bit će dokirani u aktivno mjesto butirilkolinesteraze korištenjem kvantno- mehaničkog dokinga [2, 3]. Sustavna pretraga konfiguracijskog prostora bit će provedena korištenjem kombinatoričkog pretražnog algoritma koji uključuje translacijske i rotacijske stupnjeve slobode. Za svaku generiranu konfiguraciju sustava izračunat će se energija korištenjem PM7 hamiltonijana te će se odrediti svi mogući lokalni minimumi. Za odabrane lokalne minimume optimizirat će se geometrija korištenjem kvantno-mehaničko/kvantno-mehaničkog pristupa unutar ONIOM formalizma te će se odrediti standardne Gibbsove energije vezanja.

The most important interactions of small molecules with cholinesterases were investigated by quantum mechanical docking. A systematic search of the configurational space was conducted using a combinatorial search algorithm [1]. The following degrees of freedom were taken into account: three translational and three rotational degrees of freedom. Small molecules were translated within the active site in steps of 1 Å and rotated with increments of 60°. For every generated configuration electronic energy calculations were performed using the PM7 Hamiltonian and the search for all local minima was carried out [2]. For the selected local minima combined quantum mechanical/quantum mechanical (QM/QM) optimizations were performed and relative standard Gibbs energies of binding were calculated. All quantum chemical calculations were done using the Gaussian 16 program package.

Quinuclidine derivatives are recognized as compounds with diverse biological and pharmacological activities. Their oxime analogues have been evaluated as antidotes for organophosphorus compounds (OPCs) poisoning. Although globally used as pesticides or misused as chemical weapons, there is still no efficient treatment for OPCs. According to our previous studies, it is advisable to perform cell-based assays for antidote candidates prior to other studies of their efficiency, especially in vivo, since many of them are excluded from advanced testing due to possible adverse effects. With that in mind, in a present study, we tested seven newly synthesized 3-hydroxyimino quinuclidinium bromides with different alkyl chain lengths (CnQNOH ; n = 8 – 16). Human hepatocyte (HepG2) cell line was used to determine their influence on cell viability and homeostasis, membrane integrity and oxidative status. We used standard methods for evaluating specific targets: activity of mitochondrial succinate dehydrogenase by the tetrazolium salt MTS, lactate dehydrogenase (LDH) leakage by fluorescent resazurin, mitochondrial membrane potential assay through TMRM signal and induction of reactive oxygen species (ROS) by fluorescent dye DCFDA, respectively. Our results showed that quinuclidinium oximes with a long side alkyl chain (C12, C14 and C16) were the most toxic to cells with IC50 values in micromolar range. Furthermore, compound with C12 alkyl chain displayed a notable time-dependent toxicity. More detailed analysis revealed that all three cytotoxic compounds triggered significant LDH release and decrease in membrane mitochondrial potential, indicating a necrotic-like impact. In concentrations corresponding to IC50 values, tested oximes also significantly induced ROS generation. Such results indicate that these oximes induced negative effects and could not be considered for further evaluation as antidotes. However, their observed influence on cells opens up a new perspective to investigate them as drugs for specific conditions and diseases.

Quaternary ammonium salts (QASs) are irreplaceable membrane- active antimicrobial agents that have been widely used for almost a century. Cetylpyridinium chloride (CPC) is one of the most potent QASs, however, recent data from the literature indicate a 2- to 4-fold decrease in CPC activity against resistant bacterial strains. Given the growing demand for effective antimicrobials, especially in times of current and future spread of infectious diseases, the number of resistant isolates is expected to increase. One plausible approach to address this problem is to structurally modify the CPC structure by adding other biologically active functional groups. Here, a series of QASs based on pyridine-4-aldoxime were synthesized, characterized, and tested for antimicrobial activity in vitro. Although we obtained several potent antiviral candidates, Py- C12Br, Py-C12, and Py-C14, these candidates had lower antibacterial activity than commercial CPC. AFM images showed damage to the cell membrane and no viable cells after the bacteria were exposed to 4xMIC of Py-C12 for 3 hours. We found that the addition of an oxime group to the pyridine backbone resulted in an unfavorable electron density distribution and cLogP values and disrupted the interaction with the QacR dimer that regulates efflux pump expression. MD simulations showed that binding of Py-C16 to QacR leads to dissociation of the dimer within 50 ns, whereas the same was not observed in the case of the QacR dimer and the QacR dimer bound to CPC. This explains the lower bioactivity of our compounds, as they are likely to induce premature expression of the efflux pumps.

Quinuclidine-based carbamates proved to be potent dual inhibitors of both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) and can therefore be considered as potential central nervous system agents [1]. To gain better understanding of the drug-biomolecular interactions, extensive molecular docking simulations of quinuclidine derivatives and cholinesterases were performed using ab initio molecular dynamics (MD) coupled with extensive machine learning protocol. Binding modes of the following 3-substituted quinuclidine compounds were investigated: 3-(N, N- dimethylcarbamoyloxy)quinuclidine and 3-(N, N- diethylcarbamoyloxy)quinuclidine, as well as their quaternary N-methyl derivatives . The dimensionality of the MD trajectories was first reduced by extracting only the necessary coordinates to describe the movement of the substrate within the enzyme's active site. Obtained trajectories were then further reduced in dimension by the 2nd-order tensor decomposition. In this reduced space, probability distributions (PD) of molecular geometries were calculated [2]. By finding all strict local maxima in PD functions, every possible binding mode of investigated compounds was determined. During the course of simulations strict local maxima plateaus in PD functions were calculated for each compound. The points at which the full configuration spaces were sampled were found by the machine learning protocol. For each molecule the minimal number of steps in MD simulations and principal components to obtain full configurational spaces was determined. For chosen Michaelis complexes QM/QM optimizations were performed and standard Gibbs energies of binding were calculated.

Cinchonidine, together with cinchonine, quinine and quinidine, is natural alkaloid isolated on industrial scale from the bark of the Cinchona tree.[1] Cinchona alkaloids and their derivatives have application as chiral resolving agents in NMR spectroscopy and as chiral stationary phases for chromatographic separation of different analytes. [2] 10, 11-dihydrocinchonidine was prepared by transfer hydrogenation of cinchonidine over a palladium on carbon catalyst using formic acid/ammonium formate as hydrogen donor.[3] A series of monosubstituted aromatic (phenyl group) and disubstituted aliphatic or aromatic (methyl, ethyl and phenyl groups) 10, 11- dihydrocinchonidine carbamates have been prepared by reaction of 10, 11-dihydrocinchonidine with appropriately substituted isocyanates or carbamoyl chlorides. The structures of all prepared carbamates were confirmed by 1D, 2D 1H and 13C NMR spectroscopy techniques. Evaluation of stability for prepared carbamates in solution was assessed by 1H NMR spectroscopy.

Full conformational analyses of N, N-disubstituted imidazole 2-aldoximes were performed by the grid search of potential energy surfaces (PES). PES were spanned by relevant torsional coordinates and the grid search was conducted using the rigid scan[1] as well as the adaptive relaxed scan.[2] Each scan was performed by the systematic variation in relevant torsional coordinates for a defined step size. These two scan algorithms differ in the type of quantum chemical calculation performed (single point calculation or molecular geometry optimization) where the geometry optimization is performed for those atoms that do not participate in the definition of a particular torsional coordinate. Additionally, in adaptive relaxed scan a fine control mechanism including a guided variable search of defined points on the N-dimensional surface was implemented. If the investigated system runs into the forbidden areas of PES due to the large step size, a guided variable search will reiterate and approach the forbidden areas with a smaller step thus insuring the smoothness in PES. PES were calculated at the semiempirical level of the theory using the PM7 method implemented in MOPAC2016.[3] All local minima in PES were determined from the generated surfaces and corresponding structures were further subjected to geometry optimization at the B3LYP-D3/6-311++G(d, p) level of the theory and clustered. The results of these conformational analyses will be compared, and the conformers relative content will be determined using the Maxwell-Boltzmann distribution at the room temperature.

Reduced space of multi-target antimicrobial activities was used as a dependent variable for estimation of Cinchona alkaloids derivatives [1] activities. A panel of various Gram- positive and Gram-negative bacteria provided activity data whose principal components were extracted by 2nd-order tensor decomposition. These principal components were regressed on the theoretically computed energy fingerprints of all compounds. Extensive machine learning procedure was applied for generation of multivariate linear regression models with linear combination of original variables as well as their higher- order polynomial terms. Regression models of antimicrobial activity in dependence on molecular dynamics data were builded and thoroughly validated by leave-one- out cross- validation technique (LOO-CV) [2]. The most optimal representation was selected on the basis of R2 values and LOO-CV mean squared error and is presented on Fig. 1.

Oximes and carbamates have a wide spectrum of bioactivities. Some oximes are considered as powerful reactivators of phosphorylated cholinesterase (antidotes for organophosphorus poisoning), [1] while some carbamates are used for inhibition of cholinesterases (protection of enzyme from phosphorylation).[2] Therefore, novel hybrid oxime carbamates have been designed to serve as carbamoylating agents (protection of enzyme) as well as oxime reactivators upon reaction. A series of N-benzylimidazole-2-aldoxime carbamates have been prepared. N-alkylation of commercially available imidazole was carried out with benzyl bromide in THF with sodium hydride as a base. N-benzylimidazole was converted to 2-carbaldehyde derivative by the reaction with n-butyllithium and DMF.[3] N-benzylimidazole-2-aldoximes were synthesized using hydroxylamine hydrochloride in solution and without solvent in mechanochemical reactions.[4] In order to synthesize desired oxime carbamates appropriate carbamoyl chlorides and isocyanates were used. Structures of compounds were deduced from FTIR, ATR, one- and twodimensional NMR spectra. Stability of compounds in aqueous solutions and in organic solvents was determined.

In attempt to find new, more potent antidotes against organophosphorus poisoning [1], series of novel, quaternary imidazole-2-aldoxime based compounds have been prepared. N-alkylation of commercially available imidazole was carried out with benzyl bromide in THF with sodium hydride as a base. N-benzylimidazole was converted to 2-carbaldehyde derivative by the reaction with n-butyllithium and DMF [2]. N-benzylimidazole-2-aldoxime was synthesized using hydroxylamine hydrochloride in solution and without solvent in mechanochemical reaction [3]. Quaternization of N-benzylimidazole-2-aldoxime with methyl iodide and benzyl bromides substituted with fluorine atoms and trifluoromethyl groups resulted in the desired novel quaternary compounds [1, 4]. Structures of compounds were deduced from FTIR, ATR, one- and two-dimensional NMR spectra. Furthermore, molecular modelling was used to predict the most probable orientations of compounds within the active site of the enzyme to determine the potency to reactivate phosphorylated human plasma butyrylcholinesterase (BChE). The optimized geometries for complexes of all prepared oximes with BChE have been proposed. Structural characteristics of an efficient oxime reactivator, differences in orientation and relative energies will be presented and discussed.

Antimicrobial activity of Cinchona alkaloids derivatives [1] was previously evaluated by using disc diffusion assay against a panel of various Gram-positive and Gram-negative bacteria. Principal components of the activity data were extracted by 2nd-order tensor decomposition and used as dependent variables for multivariate linear regression, whereas theoretically computed energy fingerprints of all compounds were used as independent variables. Potential energy surfaces (PES) of compounds were sampled by performing molecular dynamics simulations and then decomposed by principal component analysis. Regression models were generated by extensive machine learning multivariate linear regression – linear combinations of original variables were used as well as their higher-order polynomial terms. Obtained models were thoroughly validated by leave-one-out cross- validation technique (LOO- CV) [2]. The optimal activity/PES model based on the adjusted and the predicted R2 values as well as LOO-CV mean squared error will be presented.

Cinchona alkaloids are natural products isolated from the bark of the Cinchona tree and alkaloids isolated on industrial scale are quinine, quinidine, cinchonine and cinchonidine. They have interesting structure which includes bicyclic aliphatic quinuclidine ring and aromatic quinoline ring connected with a chiral carbon atom having hydroxyl group [1]. These alkaloids have diverse chemical applications, for example they are used as chiral resolving agents or chiral stationary phases for chromatographic separations as well as chiral catalysts or chiral ligands in asymmetric synthesis [2, 3]. In this study, transfer hydrogenation of cinchonidine and cinchonine over a palladium on carbon catalyst using formic acid/ammonium formate as hydrogen donor was studied and the course of reaction was monitored by 1H NMR spectroscopy. Since the peaks of NMR spectra of cinchona alkaloids are found in three distinct regions (aromatic, olefinic and aliphatic), monitoring of reaction was convenient to realize with 1H NMR spectroscopy. The structure of products of hydrogenation reactions was completely verified with 2D NMR spectroscopy techniques such as COSY, HSQC and HMBC and compared with unmodified alkaloids.

U okviru ovog rada opisana je priprava supstituiranih imidazolnih i benzimidazolnih derivata kako bi se dobili sintoni novih fizikalno-kemijskih svojstava i bioaktivnosti. Pripravljeni su N-benzilni, N-metilni te N-but- 3-enilni derivati heterocikla.1 Sljedila je reakcija karbonilacije N-supstituiranih derivata uz nastanak 2-karbaldehida imidazola i benzimidazola.2 Dodatno, izolirani su i analizirani nusprodukti u reakciji N- metilbenzimidazola. Osim željenog produkta, u ovoj reakciji nastaju i 2-butil-1- metilbenzimidazol i 1, 1'-dimetil-2, 2'- bibenzimidazol. Njihov nastanak objašnjen je reakcijom analognoj Čičibabinovoj aminaciji te je predožen mehanizam reakcija. Konačno, pripravljeni karbaldehidi reducirani su u odgovarajuće karbinole, 3 te je sintetiziran 2- aminometilni derivat u reakciji karbaldehida i benzilamina. Identifikacija i kontrola čistoće pripravljenih spojeva ispitana je klasičnim analitičkim postupcima te IR i NMR (1H i 13C) spektroskopijom. Pripravljeni karbaldehidi prekursori su za sintezu odgovarajućih oksima, potencijalnih antidota pri otrovanju organofosfornim spojevima te antimikrobnih agensa.2, 4 Rad je izrađen u okviru projekata Hrvatske zaklade za znanost: "Projekt razvoja karijera mladih istraživača - izobrazba novih doktora znanosti“ ESF-DOK-01-2018 te "Aktivnošću i in silico usmjeren dizajn malih bioaktivnih molekula" (IP-2016-06-3775).

Quinuclidine, bicyclic alkaloid, easily forms quaternary ammonium compounds (QACs) which exhibit diverse biological and pharmacological activity. Given their amphiphilic nature, cationic part of salts electrostatically interacts with the negatively charged phospholipids of the bacterial cell wall so that long alkyl tails of salts can penetrate into the membrane of bacteria. [1] In order to explore antimicrobial activity we have synthesized quaternary 3- hydroxyquinuclidinium salts with different lengths of alkly chains (C10, C12 and C14). Compounds were acquired in significant yields and their structures were affirmed by 1H NMR, 13C NMR and IR spectral data. The antimicrobial activity was tested against Gram-positive, Gram-negative bacteria both ATCC and clinical isolates as well against fungal strains, using broth microdiluton method to determine minimum inhibitory concentrations (MICs). Activity toward inhibition of the biofilm structures was also tested. Our preliminary results indicate that the tested compounds have a better activity against Gram-positive bacteria and the compound displaying the highest antibacterial activity is the one with the longest alkyl chain.